Pharmaceutical compositions containing pyrylium compounds, pyrylium salts and process for manufacturing a medicament containing the aforesaid compounds

Abstract: The present invention provides pharmaceutical compositions containing a pyrylium compound, a thiopyrylium compound, a selenopyrylium compound or a telluropyrylium compound or a salt of any of the aforesaid compounds as active ingredient. The compounds are selectively absorbed by cancer cells or similar growths in the human or animal body and can be used to bring about distruction of the unwanted growth on irradiation with light of wavelength 600nm to 1000nm. The invention also provides a method for the treatment of the human or animal body which comprises administering the compound to a human or animal and irradiating a locus in said animal where the compound is absorbed in order to kill cells at that locus. It further comprises the use of the aforesaid compound for the making of a medicament for use in the treatment of cancer in humans and animals.

Description: BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition containing a pyrylium compound, thiopyrylium compound, selenopyrylium compound or telluropyrylium compound (hereinafter referred to as "a pyrylium compound") or a salt of any of the aforesaid compounds as active ingredient. It also provides a method for the treatment of the human and animal body which comprises administering the compound to a human or animal and irradiating a locus in said animal where the compound is absorbed in order to kill cells at that locus. It further comprises the use of the aforesaid compound for making a medicament for use in the treatment of cancer in humans and animals. RELATED ART In the pharmaceutical field, one of the most important problems is to find medicaments for use in the treatment of cancer. One approach to the treatment of cancer is to use photochemistry. Extensive research has been carried out into the photochemical treatment of cancer, and it has been put into clinical use since 1976. There is an extensive journal and patent literature concerning photochemical treatments for cancer, see for example the general remarks of Michael J. Manya (J. Clin. Oncology, vol. 6, 1988, pp. 380-391). In these literature and patent references almost all the medicaments which have been adminstered to enable photochemical treatment to be carried out contain a porphyrin compound, particularly hematoporphyrin derivatives (HPD) or diamatoporphyrin derivatives (DHE) which have been applied clinically, and a medicament called PHOTOFRIN which contains DHE is expected to be marketed shortly by LEDERLE JAPAN Co. Ltd. The way in which photochemical treatment is carried out using a medicament containing a porphyrin-type compound will now be explained. When this medicament is given to a patient suffering from cancer, then almost all the active material is metabolised by normal cells in a few days (48 to 72 hours). However, medicament absorbed by cancer cells is not metabolised, and the amount thereof that builds up in the cell is from several times to several tens of times that in a normal cell. Then the cancer cells and surrounding tissue is irradiated with light of frequency 600-700nm, and the cancer cells in which the medicament has built up die preferentially whilst normal cells are generally unaffected by the radiation. Accordingly, it is possible to treat cancer in this way without producing unacceptable side effects. The reason why the medicament remains preferentially in the cancer cells is not clear, but it is believed to be the result of a difference in blood circulation between the cancer cells and normal cells. It is also not clear why cancer cells in which the medicament is present die when subjected to irradiation. However, it is believed that radiant energy absorbed by the medicament brings about transformation of oxygen in the vicinity of the cancer cells into singlet oxygen which has strong cytotoxicity. The photochemical method of treatment has some problems, one of which is the mismatch of the wavelength at which the porphyrin-type compound absorbs light and the wavelength of the radiation which can be used. For irradiating cells it is desirable to use radiation having a wavelength from 600nm to near infrared. The first reason is that it is necessary for the radiation to penetrate into the living tissue. For example, in the case of a skin cancer the radiation should penetrate the skin to a depth of several millimetres. Radiation of 600nm wavelength or less is absorbed or scattered by substances in the living body and does not penetrate to the desired depth. The second reason is that it is necessary to avoid any radiation damage to the hemoglobin in red blood corpuscles. Oxidised hemoglobin has absorption peaks at 540nm and 577nm and reduced hemoglobin has an absorption peak at 555nm. The energy of the radiation used for irradiation treatment is so strong that when a living body is irradiated with light of wavelength 500-600nm, hemoglobin is damaged. Furthermore, it is undesirable to use for treatment radiation having a wavelength of 1200nm or above because that radiation brings about heating of the living tissue. For the above reasons it is apparent that it is necessary to use for treatment radiation having a wavelength of 600-1000nm. However, DHE has a maximum absorption peak at 363nm and at wavelengths of 600nm or above, DHE has an absorption of only 2-3% of the peak (ε 630nm = 3.6 x 10 3 l/mol.cm; W. Roberts et al, J. Natl. Cancer Inst., vol. 80, 1988, pp. 330-336). Therefore the absorption of the light used for treatment is inefficient and it is necessary to increase the amount of medicament to be administered or to increase the intensity of the irradiating light. This requires expensive apparatus for the treatment, and give rise to harmful after-effects. Various investigations have been made with the object of overcoming these problems. For example, Japanese laid open Patent Application JP-A-1-275528 discloses porphyrin-type compounds which have been modified to extend their absorption wavelength and to increase their strength of absorption. The compound exhibiting the strongest absorption has an extinction coefficient ε of 2.24 x 10 4 l/mol.cm at 630nm wavelength. This patent also discloses a further type of compounds whose absorption strength is stronger However, it does not disclose that these compounds exhibit anti-tumour characteristics. In a paper written by W. Roberts a method is disclosed for synthesising a compound having an absorption peak with an extinction coefficient of 3.8 x 10 4 l/mol.cm at 630nm wavelength. However, in order to improve the efficiency of the treatment, a medicament having an absorption peak at longer wavelengths and having a greater extinction coefficient is desired. According to Nikkan Kogyo Shimbun on 7th June 1993, further research on MONOASPARTYLCHLORIN by Nippon Sekiyu Kagaku K.K., on PHEOPHORBIDE by Hamari Yakuhin Kogyo K.K., on ZINCFLIN by Asahi Kasei Kogyo K.K. were reported. However, these compounds are still in the research stage, and almost all the investigations now being carried out concern porphyrin-type compounds whose mode of action on cancer cells is as described above. Titanium dioxide has been investigated as an anti-cancer medicament whose mode of action on cancer cells differs from medicaments based on porphyrin-type compounds. However, in this approach there is a difficulty in transporting the titanium dioxide selectively to the cancer cells and ultraviolet radiation is required for treatment. SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a new medicament which can be used in the photochemical treatment of cancer, or similar growths that occur in the body at one or more definable places. According to the invention, there is provided a pharmaceutical composition comprising as active ingredient a therapeutically effective quantity of pyrylium compound represented by the formula [VII], wherein X is O or S, and Y - is an anion, and examples of the anion include BF 4 - , a perchlorate ion, HO 3 SCH 2 COO - , halogen ions such as a chlorine ion, bromine ion, iodine ion and fluorine ion, compounds having an anionic function such as aliphatic hdyrocarbons and aromatic sulfonates, and complex ions of transition metals such as Zn, Ni, Cu, Pt, Co and Pd. As described above, compounds which can be used as active ingredient of a medicament for the photochemical treatment of cancer according to the present invention are salts of pyrylium-type compounds. Such pyrylium-type compounds have been investigated since the beginning of the twentieth century. Early research was mainly concerned with methods for synthesing these compounds and characterising them as a dye. However, their electron transfer properties have recently attracted attention, and these compounds have been investigated as sensitisers in electro- and photoconductive compositions. There have also been some investigations of pyrylium-type compounds as anti-cancer agents. For example, in Japanese published Patent Application No. 64-52715, dimethylaminophenyl-pyridyl-thiopyrylium salt which has been substituted at the 2, 4 and 6 positions has been disclosed as an anti-cancer agent. Furthermore, in Japanese laid open Application No. 1-153683 there has been disclosed as an anti-cancer agent a 2,4,6-triphenylpyrylium or a 2,4,6-triphenyl thiopyrylium salt in which at least two hydrogen atoms of the phenyl group are substituted by dialkylamino groups. Japanese laid open Patent Application No. 1-153683 discloses compounds of the same general formula as in the present invention, and the synthesis of compounds of this type had already been reported by R. Wizinger et al (Helv. Chim. Acta., vol. 39, 5, 1965). However, none of these patent applications discloses that pyrylium compounds exhibit anti-cancer activity on selective absorption into living tissue and irradiation with light. The disclosure of EP-A-0603783 and Nucleic Acid Symposium Serie (1995), Vol. 29, p. 83-84 are similar. Typical examples of compounds which can be used as the active ingredient in a medicament for the photochemical treatment of cancer according to the present invention are shown in Table 1 below. In addition, the light absorption characteristics of typical compounds selected from Table 1 are shown in Table 2. The compound 2 shown in Table 2 has its own absorption peak in the region 600nm or above. Furthermore, compound 1 in Table 2 has an absorption peak in the region below 600nm, but it has sufficiently strong absorption that it still provides better treatment efficiency at wavelengths greater than 600nm. The mechanism by which the present pyrylium compounds attack cancer cells on exposure to light is not clear. However, the inventors have discovered that these compounds stain double stranded nucleic acid and it has therefore been postulated that the operation of the present pyrylium compounds involves attack on double stranded nucleic acid. It is therefore believed that the compounds of the invention when used for the photochemical treatment of cancer work by a different mechanism from that of the known porphyrin compounds. It is therefore possible that medicaments according to the invention will be effective for cancers which are different from those in which the porphyrin-containing medicament is effective. A variety of methods may be used for administering the medicament of the present invention to living human or animal bodies. One such method is by injection in which case the medicament is formulated as an injectable solution in a sterile physiologically acceptable solvent. Suitable solvents include balanced saline solution, an aqueous buffer solution, a water-miscible organic solvent which can dissolve the compound of the invention such as dimethyl sulfoxide or ethanol, and a mixture of the aforesaid water miscible solvent, and a mixture of water containing dissolved materials. In the case of such a mixed solution, the ratio between the aqueous and the organic portions will be selected depending on the solubility properties of the compound of the invention and/or the place and manner in which the medicament is to be administered, and so that the injection maintains its characteristics and safety. For example there may be used a mixed solution in which the water component is mixed with 5-50% by volume of water miscible organic solvent. The medicament may be injected into a vein, an artery, into the abdominal cavity, through the skin or direct into a tumour. In addition, where the injectable material contains only a water miscible organic material as solvent, the injection is preferably carried out in portions of the body other than a vein or an artery. The content of the compound of the invention in the injectable solution can be selected depending upon the solubility of the compound, the method of injection, the place where the injection is to be administered and the amount of the compound to be given. For example, the content of the active compound may be in the range of from about 0.1mg/ml to 10mg/ml. Furthermore, the amount of medicament to be administered can be selected appropriately depending upon the conditions of the patient and the disease to be treated. Some forms of the medicament according to the invention can also be given orally. Medicine for oral administration may be provided in known forms, for example in the forms of solutions, syrups, pills, capsules etc., using a suitable base material, solid diluent e.g. for making tablets, powders or capsules, or solvent for making syrups and elixirs. Medicaments for oral administration can be made by known methods. The content of the compound of the invention in a medicament for oral administration will be selected depending upon the patient to be treated and the disease to be treated and may, for example, be typically from 1-50 wt. %, particularly 5-20 wt. %. The active ingredients in the above compositions may be selected from the compounds listed in Table 1 below. The properties of some of the aforesaid compounds are as set out in Table 2 below. Compound No. Absorption (λ max ) Peak Molar absorption coefficient (ε × 10 -4 l/mol·cm) Under no existence of DNA Under existence of DNA ε 1 540nm 580nm 8.46 2 580nm 620nm 4.95 The invention will be further described by way of example with reference to the following Examples. SYNTHESIS EXAMPLE 1 100 ml of acetic anhydride was mixed with 30ml of concentrated sulfuric acid with cooling, and the resulting mixture was then heated for three hours on a water bath, while maintaining it at 80°C. Next, 20ml of acetic anhydride and 30ml of p-dimethylamino acetophenone were added thereto at room temperature, and the temperature of the solution was then raised up to 45°C, followed by stirring for 24 hours to carry out reaction, Afterward, ethanol was added to the reacted solution, the amount of ethanol being equal to that of the reacted solution. This solution was cooled, and an aqueous pottasium iodide solution was then added thereto to precipitate crude crystals. Next, the crystals were collected by filtration, and then recrystallised from a mixture of ethanol and ether (a volume ratio = 1:4) to obtain green crystals of 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl) pyrylium iodide (Compound No. 1 in Table 1 wherein Y is I). Analytical results of the obtained Compound No. 1 (Y=I) melting point = 254-257°C UV/Visual (CH 3 CN εx10 -4 ) λ max = 444nm (2.43), 550nm (8.24) NMR ( 1 H, DMSO) δ ppm: 8.3737 (1H, s) 8.2729 (1H d, J=9.0 Hz), 8.1795 (1H, d, J=9.0 Hz), 7.8864 (1H, s), 6.9117 (4H, t, J AB =J BC =9.77), 3.1829 (6H, s), 3.1340 (6H, s), 2.6809 (3H, s) FAB mass m/z 333 IR (KBr) V cm -1 : 1645, 1610 (sh), 1580 (s), 1490 (s), 1270, 1200, 1160 Furthermore, the same procedure as described above was carried out except that the aqueous potassium iodide solution was replaced with an aqueous perchloric acid solution, to obtain 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl)pyrylium perchlorate (compound 1 in Table 1 wherein Y is CIO 4 ). SYNTHESIS EXAMPLE 2 20g of sodium sulfide nonahydrate was dissolved in ionexchanged water so as to bring the total volume to 50ml. Next, 7g of sodium hydrocarbonate was added to and dissolved in this solution, and 50ml of ethanol was further added thereto under ice cooling, followed by stirring at room temperature for 30 minutes. The precipitated sodium carbonate was collected by filtration, and then washed with 25ml of ethanol. Afterward, the filtrate was mixed with the washing liquid to obtain about 125ml of the water-ethanol solution of sodium hydrogen sulfide. Next, 0.92g of 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl)pyrylium iodide obtained in Synthesis Example 1 was dissolved in 20ml of DMSO, and 5ml of the previously prepared water-ethanol solution of sodium hydrogen sulfide was then added to the resulting solution, followed by stirring at room temperature for 5 minutes. After the stirring, 0.75ml of hydoriodic acid was added thereto, and the solution was further stirred for 5 minutes. Subsequently, dichloromethane extraction and silica gel column purification were carried out in usual manner, followed by recrystallication in an ethanol-ether mixed solution (a volume ratio = 1:4), to obtain the crystals of 2-methyl-4,6-bis-(4-N,N-dimethylaminopheynl)thiopyrylium iodide (Compound No. 2 in Table 1 wherein Y is I). Analytical results of the obtained Compound No. 2 = (Y=1): Melting point: 246-248°C UV/visual (CH 3 CN ε x 10 -4 ) λmax: 495nm (2.50), 587nm (4.95) NMR ( 1 H, DMSO) δ ppm: 8.5679 (1H, s), 8.84323 (1H, s), 8.2436 (2H, d, J=9.27 Hz), 7.9786 (2H, d, J=9.28), 6.8959 (4H, t, J AB =J BC =9.28), 3.1756 (6H, s), 3.1157 (6H, s), 2.8323 (3H, s) FAB mass m/z 349 IR (KBr) ϑ cm -1 :1600 (s), 1560 (s), 1460 (s), 1370 (s), 1260 (s), 1160 (s) Furthermore, the same procedure as dsecribed above was carried out except that hydoriodic acid was replaced with an aqueous perchloric acid solution, to obtain 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl)-thiopyrylium perchlorate (Compound No. 2 in Table 1 wherein Y is CIO 4 ). EXAMPLE 1 L-929 cells which are typical cultured cancer cells from mice were planted onto a culture medium containing E-MEM + 10% FCS. The density of the cells in the medium was 10 6 cells/dish (20ml). Thereafter, the cells were maintained at 37°C and cultured for 36 hours. Four such cultures using the same medium were prepared. Then compound 1 (Y=I) was added to each of the above cultured media and the concentration of the compound 1 was varied from sample to sample as shown in Table 3. Then radiation of wavelength from 560-610nm to which the compound 1 is responsive was supplied using a fluorescence microscope for the interval of time indicated in Table 3. Thereafter, each medium was cultured for 12 hours, after which the cells were observed using a microscope. The percentage of the cells which had died in each sample was estimated and is shown in Table 3. As is apparent from this table, the compound 1 in a concentration in the medium of 0.1-0.5 µg/ml is effective to kill cancer cells on irradiation for a period of 1-5 minutes. It has been found that uptake of compound 1 by the cancer cells is rapid. Furthermore, cancer cells which were treated by the compound of Example 1 and not irradiated, and cancer cells which were irradiated without addition of the medicament were not killed and continued to grow in culture under the test conditions. Irradiated Time Concentration of the Compound 1 (µg /ml) 0.05 0.1 0.25 0.5 5 (min) 20~30 (%) 100 (%) 100 (%) 100 (%) 3 - 70 ~ 80 ~ 100 ~ 100 1 - - 50 ~ 60 ~ 90 EXAMPLE 2 The same test as in Example 1 was carried out except that the active compound used was compound 2 (Y=I) instead of compound 1. The amount of compound 2 which was administered to the cells is shown in Table 4 and the percentage of cells which died for the various irradiation times is also indicated. The light used to irradiate the cells at a wavelength of 600-650nm. The results shown in Table 4 indicate that compound 2 has a greater killing effect than compound 1 against cancer cells. It was also found that cancer cells not subjected to compound 2 and irradiated, and such cells which had been subjected to the compound but not irradiated were not killed and continued to grow in culture. Irradiated Time Concentration of the Compound 2 (µg/ml) 0.025 0.05 0.075 0.1 5 (min) ~ 50 (%) 100 (%) 100 (%) 100 (%) 3 - 80 100 100 1 - ~ 10 80 100 Examples of Medicaments EXAMPLE 3 The compound 1 (Y=I) was dissolved at a concentration of 1mg/ml into balanced saline solution to give an injectable solution. EXAMPLE 4 The compound 2 (Y=I) was dissolved into a mixed solution containing 80 volume % of balanced saline solution and 20 volume % of ethanol to give an injectable solution. The concentration was Smg/ml. Acute Toxicity Test EXAMPLE 5 The compound 1 (Y=I) was dissolved into a balanced saline solution and was injected into each of 2 nude mice of weight 200-400g through a vein in the leg of the mouse. The amount of solution injected was 1mg to 1kg of the weight of each mouse. Subsequent observation of the mice indicated no adverse effects, and they remained in satisfactory health after the injection. EXAMPLE 6 The toxicity test of Example 5 was repeated except that the compound 2 was used in place of the compound 1. Again, observation of the mice indicated no ill effects, and the mice remained in good health after the injection. EXAMPLE 7 The test of Example 5 was repeated except that the compound 1 (Y=I) was dissolved in dimethyl sulfoxide. The amount of solution injected was lmg per 1kg of the mouse's weight. Subsequent injections on further groups of mice were carried out at 5mg per 1kg of the mouse's weight and 25mg per 1kg of the mouse's weight. No adverse effect on the mice was observed, and they continued to be in good health after the injection. EXAMPLE 8 The same test as in Example 7 was carried out except that the active ingredient was compound 2 (Y=I) instead of compiund 1. Again, no ill effects were observed in the mice who continued to thrive after the injection. The results of the tests in Examples 5 to 8 indicate that the compounds tested have low acute toxicity. Treatment of Cancer Cells in a Living Body EXAMPLE 9 Ten nude mice having weight 200-400g were selected, and they were separated into five groups referred to as A, B, C, D and E, each containing two nude mice. Each of the mice was inocculated on its back with KATO III, which is a culture of stomach cancer cells from a human patient. The cancer cells were inocculated into the central part of the back of each mouse, after which the mice were kept for ten days. It was found that the cancer in each mouse had grown to a size of about 5mm width, about 5mm length and about 3mm height. The mice in groups A, B and E were injected with the compound 1 (Y=I) in balanced saline solution through a vein in the leg. The amount of compound 1 which was administered to each mouse was lmg per 1kg of the mouse's weight. Thereafter, the cancerous growth in each mouse in the group A and C was irradiated with white light in which the radiation of wavelength 400nm or less and radiation of wavelength 1000nm or above was filtered out. In group E, a part of every mouse's back was irradiated with the white light. In each case the amount of irradiation was 50J. Thereafter, the cancerous region of each mouse in the group A to D was observed and the skin of each mouse in the group E was observed. The results are as shown in Table 6. EXAMPLE 10 The same test as in Example 9 was carried out except that the compound 2 was used in place of the compound 1. The results are shown in Table 7. The results given in Tables 6 and 7 indicate that compound 1 and compound 2 are effective in the photochemical treatment of human cancer cells growing in live mice. However, when the mice in group E or E' were irradiated, no change was observed so that it was apparent that the affinity of compound 1 and compound 2 for the cancer cells was different from the affinity of compound 1 and compound 2 for normal cells. The above results suggest that cancer cells can be treated with a medicament as set out herein, which is of completely different structure from known porphyrin-type photochemical medicaments. The treatment has been found to be particularly effective using pyrylium-type compounds having an absorption maximum in the range 600-1000nm and having strong absorption strength. Furthermore, the salts of the present pyrylium-type compounds are readily absorbed by cells of the living body, including in particular some types at least of cancer cell so that the treatment can be carried out effectively. A selection of the substituent groups enables the absorption peak wavelength to be adjusted within the range 600-1000nm so that the wavelength of the radiation required for treatment can also be selected.

Claims: A pharmaceutical composition comprising as active ingredient a therapeutically effective quantity of pyrylium compound represented by the formula [VII], (wherein X is O or S, and Y - is an anion). The composition of claim 1, wherein the active ingredient is a pyrylium compound of formula (VII) in which Y - is CIO 4 - or I - . The composition of claim 1 or claim 2, wherein the active ingredient is a 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl) pyrylium salt. The composition of claim 1 or claim 2, wherein the active ingredient is a 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl)thiopyrylium salt. The composition of any of claims 1 to 4 in the form of an injectable solution. The composition of any of claims 1 to 5, wherein there is present a solid diluent. The composition of any of claims 1 to 4 in the form of a syrup or elixir. Use of a compound as defined in any of claims 1 to 4 for the manufacture of a medicament for the treatment of cancer. Use of a compound as defined in any of claims 1 to 4 for the manufacture of a medicament for the photo-dynamic treatment of cancer.

Inventors: KAWAGUCHI MASAHIRO, OKAMOTO TADASHI, YAMAMOTO NOBUKO, KAWAGUCHI, MASAHIRO, OKAMOTO, TADASHI, YAMAMOTO, NOBUKO, KAWAGUCHI, MASAHIRO, C/O CANON KABUSHIKI KAISHA, OKAMOTO, TADASHI, C/O CANON KABUSHIKI KAISHA, YAMAMOTO, NOBUKO, C/O CANON KABUSHIKI KAISHA
Applicants: CANON KK, CANON KABUSHIKI KAISHA
Application year: 1994
Main classification: A61K31
IPC codes: A61K31, C07D345, C07D335, A61K41, C07D309, A61P43, A61P35